CLSY1 Antibody
Description
Molecular Function of CLSY1
CLSY1 is an SNF2-family ATP-dependent chromatin remodeler that facilitates locus-specific siRNA production and DNA methylation . Key functions include:
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RdDM Pathway Integration: Partners with Pol IV and SHH1 to direct 24nt-siRNA synthesis .
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Chromatin Remodeling: Mediates nucleosome repositioning to allow Pol IV access to heterochromatic regions .
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Dual Regulatory Role: Balances DNA methylation and demethylation at specific genomic loci .
Table 1: CLSY1 Functional Characteristics
Antibody Applications in CLSY1 Research
CLSY1 antibodies are primarily used for:
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Co-immunoprecipitation (Co-IP): Identifying CLSY1 interaction partners (e.g., Pol IV, SHH1, DTF1) .
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Chromatin Immunoprecipitation (ChIP): Mapping CLSY1 binding sites at RdDM target loci .
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Mutant Validation: Confirming CLSY1 protein loss in clsy1 knockout lines .
Table 2: Experimental Systems Using CLSY1 Antibodies
CLSY1-Pol IV Interaction Mechanism
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Docking Motif: CLSY1 binds Pol IV through NRPD1’s CYC-YPMF domain, essential for siRNA biogenesis .
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Competitive Binding: All four CLSY proteins (CLSY1-4) compete for this docking site, ensuring locus specificity .
Epigenetic Regulation Dynamics
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Methylation-Demethylation Balance: CLSY1 mutants show hyper-methylation at loci regulated by CLSY2-4, indicating compensatory demethylation .
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Nucleosome Remodeling: CLSY1 facilitates Pol IV access by reducing nucleosome density at target sites .
Interaction Network
CLSY1 operates within a protein network critical for RdDM:
Table 3: CLSY1-Associated Proteins
| Protein | Role in RdDM | Interaction Evidence | Citation |
|---|---|---|---|
| NRPD1 | Pol IV catalytic subunit | Co-IP, yeast two-hybrid | |
| SHH1 | Binds H3K9me2 marks | IP-MS of Pol IV complexes | |
| DTF1 | Chromatin recruiter | Co-IP with CLSY1 and Pol IV |
Technical Considerations
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Antibody Specificity: Most studies use epitope-tagged CLSY1 (e.g., Myc, HA) due to lack of commercial antibodies .
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Mutant Lines: clsy1 alleles (e.g., clsy1-1 to clsy1-6) show siRNA deficits and DNA methylation defects .
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Limitations: Functional redundancy among CLSY1-4 necessitates high-order mutants for phenotypic analysis .
Product Specs
**Constituents:** 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- A protein containing an SNF2 domain (CLASSY1) has been identified. It collaborates with RDR2 and NRPD1a in the propagation of transgene silencing and the production of endogenous 24-nucleotide short interfering RNAs (siRNAs). [CLSY1] PMID: 17526749
Q&A
Experimental Design for CLSY1 Antibody Use
Q: How should I design an experiment to study the role of CLSY1 using a CLSY1 antibody in plant research? A: To study CLSY1, you can use co-immunoprecipitation (co-IP) experiments to identify proteins that interact with CLSY1. This involves expressing a tagged version of CLSY1 (e.g., CLSY1-Myc) and then using an antibody against the tag to pull down associated proteins for mass spectrometry analysis . Additionally, Western blotting can be used to assess protein levels in mutant versus wild-type plants.
Data Interpretation and Contradiction Analysis
Q: How do I analyze and reconcile contradictory data from different studies using CLSY1 antibodies? A: When analyzing contradictory data, consider the experimental conditions, antibody specificity, and sample preparation. For instance, differences in antibody specificity or experimental conditions (e.g., tissue type, developmental stage) might lead to varying results. Use controls and validate findings with multiple methods (e.g., co-IP, Western blot) to ensure consistency.
Advanced Research Questions: CLSY1 Interactions
Q: What are the known interactions of CLSY1 with other proteins, and how can these be studied using CLSY1 antibodies? A: CLSY1 is known to interact with Pol IV and SHH1 in plants . These interactions can be studied using co-IP followed by mass spectrometry or Western blotting. For example, expressing a tagged version of CLSY1 and then using an antibody against the tag to co-purify associated proteins can help identify these interactions.
Methodological Considerations for Antibody Selection
Q: What factors should I consider when selecting a CLSY1 antibody for my research? A: When selecting a CLSY1 antibody, consider the species specificity (e.g., Arabidopsis thaliana), the type of assay (e.g., Western blot, co-IP), and the epitope specificity. Ensure the antibody is validated for your specific application and has been tested in similar experimental conditions to minimize cross-reactivity issues.
Troubleshooting Common Issues
Q: How can I troubleshoot common issues with CLSY1 antibody experiments, such as low signal or non-specific binding? A: Common issues like low signal or non-specific binding can be addressed by optimizing antibody concentrations, using appropriate blocking agents, and ensuring proper sample preparation. Additionally, using controls (e.g., negative controls without the primary antibody) can help identify non-specific binding.
Integration with Other Techniques
Q: How can CLSY1 antibodies be integrated with other techniques (e.g., RNA sequencing, ChIP-seq) to enhance research outcomes? A: CLSY1 antibodies can be used in conjunction with techniques like ChIP-seq to study chromatin modifications associated with CLSY1. For example, after co-IP with a CLSY1 antibody, the precipitated chromatin can be analyzed by ChIP-seq to identify genomic regions bound by CLSY1.
